Tetanus toxin is a protein neurotoxin of 1315 amino acids. The mechanisms by which tetanus toxin binds to and enters cells, and inhibits neurosecretion are not well understood. Hc (previously referred to as Fragment C) is a about 50,000 M.W. peptide derived by proteolysis from the carboxyl terminus of tetanus toxin that retains the ability to bind to gangliosides present in neuronal cell membranes. We have previously prepared a number of deletion mutants of Hc and identified the carboxyl terminal 5 amino acids as a region critical for binding to purified ganglioside. To determine if therequirements of Hc binding to purified ganglioside are similar to those for binding to target cells, these mutant forms of Hc have been examined for the ability to bind to spinal cord neuronal cultures. The binding of these proteins to neuronal cells paralleled the binding to purified ganglioside. Mutants with up to 263 amino acids deleted from the amino terminus retained ganglioside binding activity. The detection of 5 amino acids from the carboxyl terminus did not effect binding while the deletion of 10 or more carboxyl terminal amino resulted in a 75-80% loss of binding relative to Hc. The deletion mutants were more sensitive than Hc to proteolysis with trypsin, suggesting alterations in conformation. The receptor binding activity of Hc was not retained in a peptide corresponding to the carboxyl terminal 20 amino acids. These data suggest the carboxyl terminal of Hc is important for maintaining a conformation necessary for binding to receptor.
